Wilson J M, Tanko Q, Wendland M M, Meany J E, Nedved J F, Pocker Y
Department of Chemistry, Seattle University, Washington 98122, USA.
Physiol Chem Phys Med NMR. 1998;30(2):149-62.
The present work demonstrates that the high-activity zinc metalloenzyme, carbonic anhydrase (CA II) from bovine erythrocytes is inhibited by the cyclic sulfimide, saccharin, and 2- and 4-carbobenzoxybenzene sulfonamide. A spectrophotometric method was employed to monitor the enzymatically catalyzed hydrolysis of p-nitrophenyl acetate by following the increase in absorbance at 410 nm which accompanies p-nitrophenoxide/p-nitrophenol formation. The more rapid enzymatic hydration of CO2 was monitored by using a stopped-flow spectrophotometer as well as by a modified colorimetric method of Wilbur and Anderson. The studies show that, at a given molar ratio of inhibitor to enzyme, the degree of inhibition of the enzymaic hydration of CO2 and hydrolysis of p-nitrophenyl acetate by the inhibitory compounds is essentially the same. Kinetic analyses were made at 25.0 degrees at pH 6.5 (MES buffers), pH 6.9 (HEPES buffers) and pH 7.9 (HEPES buffers) with ionic strength regulated by the addition of appropriate quantities of sodium sulfate. Lineweaver-Burk plots were used to evaluate apparent inhibition constants for each of the three inhibitors. For all the inhibitors studied, inhibition appears to be mixed (competitive/noncompetitive). For saccharin in the presence of sodium sulfate, the extent of inhibition is considerably decreased. It was found for the three inhibitors that the inhibitory potency decreases with increasing pH, and that the inhibitory potency is extremely sensitive to the shape of these rather closely related molecules. For example, apparent inhibition constants for the enzymatic hydrolysis of p-nitrophenyl acetate at pH 6.9 were Ki (saccharin) = 0.20 mM, Ki (2-carbobenzoxybenzene sulfonamide) = 0.54 mM and Ki (4-carbobenzoxybenzene sulfonamide) = 1.6 microM. For the enzymatic hydration of CO2 at pH 6.9, 0.10 mM saccharin caused 50% inhibition while 7.0 nM 4-carbobenzoxybenzene sulfonamide resulted in 50% inhibition. The results suggest that sulfonamide inhibition is caused by formation of a monodentate ligand at the zinc ion of the enzyme active site and that the more linear 4-carbobenzoxybenzene sulfonamide is better able to enter a conical enzyme active site than is 2-carbobenzoxybenzene sulfonamide or saccharin.
目前的研究表明，来自牛红细胞的高活性锌金属酶——碳酸酐酶（CA II）受到环状磺酰亚胺、糖精以及2-和4-苄氧羰基苯磺酰胺的抑制。采用分光光度法，通过跟踪410 nm处吸光度的增加来监测对硝基苯乙酸的酶促水解，该吸光度的增加伴随着对硝基苯氧负离子/对硝基苯酚的形成。使用停流分光光度计以及Wilbur和Anderson改进的比色法监测CO2更快的酶促水合作用。研究表明，在给定的抑制剂与酶的摩尔比下，抑制性化合物对CO2酶促水合作用和对硝基苯乙酸水解的抑制程度基本相同。在25.0℃、pH 6.5（MES缓冲液）、pH 6.9（HEPES缓冲液）和pH 7.9（HEPES缓冲液）下进行动力学分析，通过添加适量的硫酸钠调节离子强度。使用Lineweaver-Burk图评估三种抑制剂各自的表观抑制常数。对于所有研究的抑制剂，抑制作用似乎是混合型的（竞争性/非竞争性）。在硫酸钠存在下，糖精的抑制程度显著降低。对于这三种抑制剂发现，抑制效力随pH升高而降低，并对这些密切相关分子的形状极为敏感。例如，在pH 6.9时，对硝基苯乙酸酶促水解的表观抑制常数为：Ki（糖精）= 0.20 mM，Ki（2-苄氧羰基苯磺酰胺）= 0.54 mM，Ki（4-苄氧羰基苯磺酰胺）= 1.6 μM。在pH 6.9时，对于CO2的酶促水合作用，0.10 mM糖精引起50%的抑制，而7.0 nM 4-苄氧羰基苯磺酰胺导致50%的抑制。结果表明，磺酰胺抑制是由于在酶活性位点的锌离子处形成单齿配体所致，并且与2-苄氧羰基苯磺酰胺或糖精相比，更呈线性的4-苄氧羰基苯磺酰胺更能进入锥形的酶活性位点。
